Drosophila Cell Culture
A. S2, S2C, S2*, S2R+, S3, Kc(167), DL1, & DL2 cells: Schneider''s/ 10% FBS/ PS M3/10% FBS/PS/Insulin Complete M3 Medium Heat Inactivating Fetal Bovine Serum
Insulin Stock (100x)
Fly Extract
Cells grow @ 23-25°C and @ RT Split one flask of cells (most recent date) into two new T-75 flasks (VWR# BD353136) when culture is confluent. Keep other flask as a backup. Monitor growth status by microscopy before splitting and decide whether to adjust recommended dilution factor accordingly. A. Semi-adherent cell lines - will stick to new flask but come loose over time
450 ml Schneider''s medium (Gibco #11720-034)
(pour off 50 ml into Falcon to save as serum-free)
***[S2R+ and Kc cells will not grow in Schneider''s medium from Sigma]
50 ml Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 ml 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
Sterile filter (0.2 µ) Store at 4°C - do not freeze
450 ml Shields and Sang M3 insect medium (Sigma #S8523)
(pour off 50 ml into Falcon to save as serum-free)
50 ml Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
5 ml 1:100 Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
20 ug/ml Insulin (Sigma #I6634)
Sterile filter (0.2 µ) Store at 4°C - do not freeze
Shields and Sang M3 insect medium (Sigma #S3652)
2% Fetal Bovine Serum (JRH #12103-78P) - Heat Inactivated (aliquoted in -20)
2.5% Fly extract (see below)
0.125 IU/ml (0.5mg/ml) Insulin (Sigma #I6634)
1x Penicillin-Streptomycin (Gibco #15070-063) (aliquoted in -20)
Sterile filter (0.2 µ)
To make 400ml extract, use 60g of frozen flies.
To make 500ml extract, use 75g of frozen flies.
Do not need CO2
Split every 3-4 days to maintain
Density sensitive - die if too dense or too dilute
S2*
3-4 days
1:3 - 1:4
S2c
3-4 days
1:3 - 1:4
Kc(167)
3-4 days
1:3 - 1:4
l(2)mbn
3-4 days
1:3 - 1:4
DL1 | 3-4 days | 1:8 (1:5 - 1:10) |
DL2 | 3-4 days | 1:8 (1:5 - 1:10) |
S3 | 3-4 days | 1:3 |
S2R+ | 3-4 days | 1:3 - 1:4 |
CL8 | 4-5 days | 1:5-1:10 |
BG2 | 4-5 days | 1:3-1:4 |
BG6 | 4-5 days | 1:3-1:4 |
SL2, S2C, S2*
Detach cells from the flask either by banging or scraping
Pipet cells up and down about 10 times to resuspend cells and separate clumps
Aliquot cells into new flasks accordingly
DL2, DL1, S2R+, Kc167, Clone 8, S3
Protocol 1:
Remove all medium
Scrape cells with scraper
Resuspend cells with 10mL of fresh medium, pipetting up and down about 10 times
Aliquot cells into new flasks accordingly
Protocol 2:
Remove medium
Wash in PBS to remove any serum
Add 5 ml trypsin; let sit 5-10''
Bang cells off flask.
Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
Spin down cells at 1200-1400 rpm for 5 min.
Resuspend cells in serum medium.
Aliquot cells into new flasks accordingly
Protocol 3:
Detach cells from the flask either by banging or scraping
Pipet cells up and down about 10 times to resuspend cells and separate clumps
Aliquot cells into new flasks accordingly
BG2
- Remove medium (save and filter through syringe filter for use as "conditioned medium").
- Wash in PBS to remove any serum
- Add 5 ml trypsin; let sit 5-10''
- Bang cells off flask.
- Add 5 ml serum medium to inactivate trypsin and wash remaining cells off bottom of flask.
- Spin down cells at 1200-1400 rpm for 5 min.
- Resuspend cells in conditioned medium.
- Aliquot cells into new flasks accordingly
Can also use Accutase instead of trypsin.
- Grow cells to subconfluencey - approximately 1-2 x 107 cells/ml.
- Label appropriate # of cryovials.
- Remove cells from flask (trypsinize and resuspend in medium if necessary).
- Spin cells at 1200 rpm 5''
- Aspirate off medium.
- Resuspend at approximately 1.1 x 107 cells/ml in freezing medium:
FBS + 10% DMSO, filtered OR Complete medium + 10% DMSO, filtered - Aliquot 1ml cell suspension/cryovial.
- Put vials in foam box, tape closed, and place in -70°C.
- After a few days, transfer vials to LN2 for long term storage.
- Prepare 15ml conical tube with 5ml medium.
- Thaw cells quickly in water bath.
- Just before cells are completely thawed, decontamiate the outside of the tube with 70% EtOH.
- Transfer the cells to the conical tube with 5ml medium.
- Spin at 1200rpm 5''.
- Aspirate and resuspend cells in 5ml medium.
- Plate in a T25 flask.
- Watch daily. Initially, cells may need to be split at irregular intervals.
- Remove ~0.5 ml cells from flask (trypsinize cells if necessary) into eppendorf.
- Dilute cells into Trypan Blue according to estimated density.
- (confluent cultures try 30:70 µl to 50:50 µl of cells:TB).
- Transfer 10 - 12 µl cells to haemocytometer.
- Count cells in opposite corners (~100-200/ large square).
- Calcuation:
(count #1 + count #2) / 2 x (dilution factor) x 104 = X cells/ml (usually ~1-10 x 107)